Acid Polysaccharide-Induced Amorphous Calcium Carbonate (ACC

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Acid Polysaccharide-Induced Amorphous Calcium Carbonate (ACC) Films: Colloidal Nanoparticle Self-Organization Process Chao Zhong† and C. Chang Chu*,†,‡ Fiber and Polymer Science Program, Department of Fiber Science and Apparel Design, Cornell UniVersity, Ithaca, New York 14853-4401, and Biomedical Engineering Program, Cornell UniVersity, Ithaca, New York 14853-4401 ReceiVed October 23, 2008. ReVised Manuscript ReceiVed January 5, 2009 Amorphous calcium carbonate (ACC) plays important roles in biomineralization, and its synthesis in vitro has been of keen interest in the field of biomimetic materials. In this report, we describe the synthesis of ACC films using a novel acid polysaccharide, maleic chitosan, as an additive. We prepared the films by directly depositing them onto TEM grids and examined them using polarized optical microscopy (POM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) combined with selected area electron diffraction (SAED). This enabled us to examine their formation and mesostructure without introducing artifacts. We observed that, in the presence of maleic chitosan, the ACC films are formed through a particle buildup process, with aggregation and coalescence occurring simultaneously. Nanoparticles with a size of less than 10 nm appear to be the basic units responsible for such selforganization. We suggest that the acid polysaccharide plays an important role in forming and stabilizing these nanoparticles, and we propose a colloidal nanoparticle self-organization model to explain the formation of the ACC films.

Introduction Amorphous calcium carbonate (ACC) is one of the six known forms of calcium carbonate. Albeit unstable in its pure form under ambient conditions, ACC, like its crystalline polymorphs, can be produced, utilized, and stabilized by many biological organisms from different taxa.1,2 It is also known that ACC has important functions in biominerals and biomineralization processes. For example, it can be used as temporary storage deposits1,3 and structural skeletal elements.1,4,5 Recent evidence also shows that ACC acts as a transient precursor to its two major crystalline polymorphs, calcite and aragonite.6-10 Biological occurrences of ACC usually involve organic macromolecules and, sometimes, magnesium and phosphate, factors that may contribute to the stabilization of ACC.2,11 Inspired by such findings, and trying to understand the biological mechanisms of ACC stabilization, several studies have synthesized ACC in the presence of additives and/or templates.12,13 For example, ACC phase with stability over periods * To whom correspondence should be addressed. Telephone: (607) 2551938. Fax: (607) 255-1093. E-mail: [email protected]. † Fiber and Polymer Science Program, Department of Fiber Science and Apparel Design. ‡ Biomedical Engineering Program.

(1) Addadi, L.; Raz, S.; Weiner, S. AdV. Mater. 2003, 15, 959–970. (2) Weiner, S.; Levi-Kalisman, Y.; Raz, S.; Addadi, L. Connect. Tissue Res. 2003, 44, 214–218. (3) Raz, S.; Testeniere, O.; Hecker, A.; Weiner, S.; Luquet, G. Biol. Bull. 2002, 203, 269–274. (4) Aizenberg, J.; Lambert, G.; Addadi, L.; Weiner, S. AdV. Mater. 1996, 8, 222–225. (5) Aizenberg, J.; Weiner, S.; Addadi, L. Connect. Tissue Res. 2003, 44, 20– 25. (6) Beniash, E.; Aizenberg, J.; Addadi, L.; Weiner, S. Proc. R. Soc. London, Ser. B 1997, 264, 461–465. (7) Raz, S.; Hamilton, P. C.; Wilt, F. H.; Weiner, S.; Addadi, L. AdV. Funct. Mater. 2003, 13, 480–486. (8) Politi, Y.; Arad, T.; Klein, E.; Weiner, S.; Addadi, L. Science 2004, 306, 1161–1164. (9) Ma, Y.; Weiner, S.; Addadi, L. AdV. Funct. Mater. 2007, 17, 2693–2700. (10) Weiss, I. M.; Tuross, N.; Addadi, L.; Weiner, S. J. Exp. Zool., Part B 2002, 293, 478–491. (11) Lam, R. S. K.; Charnock, J. M.; Lennie, A.; Meldrum, F. C. CrystEngComm 2007, 9, 1226–1236. (12) Han, T. Y. J.; Aizenberg, J. Chem. Mater. 2008, 20, 1064–1068.

ranging from hours to months has been achieved by the addition of magnesium,14,15 self-assembled monolayers (SAMs),12,16,17 biological macromolecules,4,5 synthetic polymers,18-23 and even small molecules.24 Stable continuous ACC or crystalline films transformed by ACC precursors are of particular interest because they provide an environmentally friendly process for fabricating ceramic films.19 Examples include the use of amphiphilic porphyrin templates coupled with poly(acrylic acid) to prepare thin continuous calcium carbonate films;25 the preparation of a polypeptide-stabilized ACC film using an unconventional crystallization process called “polymer-induced liquid precursor” (PILP);20 and the fabrication of a millimeter-sized single calcite film via the transformation of metastable ACC films on micropatterned SAMs.16 We are interested in understanding the stabilization of ACC in biological systems, and although there are reports using synthetic polymers to stabilize ACC phase, most of them do not use polymers that have direct biological relevance. Accordingly, we chose to use a synthetic acid polysaccharide, maleic chitosan (a derivative of chitin, Supporting Information Figure S1), as an (13) Faatz, M.; Grohn, F.; Wegner, G. AdV. Mater. 2004, 16, 996–1000. (14) Loste, E.; Wilson, R. M.; Seshadri, R.; Meldrum, F. C. J. Cryst. Growth 2003, 254, 206–218. (15) Ajikumar, P. K.; Wong, L. G.; Subramanyam, G.; Lakshminarayanan, R.; Valiyaveettil, S. Cryst. Growth Des. 2005, 5, 1129–1134. (16) Aizenberg, J.; Muller, D. A.; Grazul, J. L.; Hamann, D. R. Science 2003, 299, 1205–1208. (17) Lee, J. R. I.; Han, T. Y.-J.; Willey, T. M.; Wang, D.; Meulenberg, R. W.; Nilsson, J.; Dove, P. M.; Terminello, L. J.; van Buuren, T.; De Yoreo, J. J. J. Am. Chem. Soc. 2007, 129, 10370–10381. (18) Xu, X. R.; Han, J. T.; Cho, K. Chem. Mater. 2004, 16, 1740–1746. (19) Gower, L. A.; Tirrell, D. A. J. Cryst. Growth 1998, 191, 153–160. (20) Gower, L. B.; Odom, D. J. J. Cryst. Growth 2000, 210, 719–734. (21) Donners, J. J. J. M.; Heywood, B. R.; Meijer, E. W.; Nolte, R. J. M.; Roman, C.; Schenning, A. P. H. J.; Sommerdijk, N. A. J. M. Chem. Commun. 2000, 1937–1938. (22) Bolze, J.; Peng, B.; Dingenouts, N.; Panine, P.; Narayanan, T.; Ballauff, M. Langmuir 2002, 18, 8364–8369. (23) Bolze, J.; Pontoni, D.; Ballauff, M.; Narayanan, T.; Colfen, H. J. Colloid Interface Sci. 2004, 277, 84–94. (24) Xu, A. W.; Yu, Q.; Dong, W. F.; Antonietti, M.; Colfen, H. AdV. Mater. 2005, 17, 2217–2221. (25) Xu, G. F.; Yao, N.; Aksay, I. A.; Groves, J. T. J. Am. Chem. Soc. 1998, 120, 11977–11985.

10.1021/la803541m CCC: $40.75  2009 American Chemical Society Published on Web 02/03/2009

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organic additive to induce ACC film formation. Acid polysaccharides rich in either carboxyl or sulfated groups have been found to control the formation and morphology of corresponding biominerals in some organisms,26-30 and it has been suggested that sugar moieties in some biogenic macromolecules are a possible factor in stabilizing ACC both in vitro and in vivo.4,31 And yet, despite their importance in the field of biomineralization, polysaccharide macromolecules have received very little attention from in vitro studies of calcium carbonate mineralization, and virtually no work has used polysaccharides to produce and stabilize ACC phase. Despite the previous work on the synthesis of ACC films, the formation and mesostructure of ACC films are still not well understood. It has widely been accepted that ACC film formation occurs when preformed polymer-induced liquid precursor (PILP) droplets (2-4 µm) wet the substrate and spread into film form.20 However, in the absence of an adequate investigation of ACC film mesostructure, we believe it is premature to accept this statement about ACC film formation. Accordingly, we set out to examine the formation and the mesostructure of ACC films using a new approach. In particular, we used maleic chitosan as an organic additive to directly induce ACC film formation onto transmission electron microscopy (TEM) grids followed by probing their mesostructure using TEM combined with selected area electron diffraction (SAED). Previous work has shown TEM to be a useful tool for studying ACC precursor structures32 and the nucleation of CaCO3 crystallization at an early stage.33 Our studies may contribute to the understanding of biological mechanisms in ACC stablization. We also believe that our studies will provide insights into the general formation mechanism of ACC films in the presence of additive/template systems.

Experimental Section ACC Film Precipitation. All chemicals were of analytical grade and used as received from Sigma. TEM grids (G300H-Cu, 300 Mesh Hex) were purchased from Electron Microscopy Sciences. All small pieces of glass coverslips were cleaned and sonicated in ethanol, rinsed with double distilled water, and dried with a constant flow of N2. The formation of amorphous CaCO3 was carried out in small Petri dishes (5 mL), which were put in a closed desiccator at room temperature (22 °C). In a typical procedure, 75 µL of 10 mg/mL maleic chitosan solution was injected into 3 mL of 50 mM CaCl2 solution (filtered by a microfilter with a pore size of 40 µm) in a 10 mL clean centrifuge tube. After being mixed homogeneously in the tube for 1 min, the solution was transferred into the 5 mL Petri dish. Next, one piece of glass slide (18 mm) or the TEM grid was carefully put into the Petri dish for collecting the amorphous phase (floating on the surface of the solution). After that, the bottles were covered with parafilm, which was punched with three needle holes, and placed in a larger desiccator. Three small glass bottles (15 mL) of crushed ammonium carbonate were also covered with parafilm punched with one needle hole in the center and placed at the bottom of the desiccator. The formation of ACC usually took place within 1 h; samples were thus collected at 30 min and 40 min. (When stored in a vacuum, ACC films can be stabilized for at least 3 months.) At (26) Ozaki, N.; Sakuda, S.; Nagasawa, H. Biochem. Biophy. Res. Commun. 2007, 357, 1172–1176. (27) Albeck, S.; Weiner, S.; Addadi, L. Chem.sEur. J. 1996, 2, 278–284. (28) Andrade, L. R.; Salgado, L. T.; Farina, M.; Pereira, M. S.; Mourao, P. A. S.; Filho, G. M. A. J. Struct. Biol. 2004, 145, 216–225. (29) Arias, J. L.; Neira-Carrillo, A.; Arias, J. I.; Escobar, C.; Bodero, M.; David, M.; Fernandez, M. S. J. Mater. Chem. 2004, 14, 2154–2160. (30) Goldberg, W. M. Tissue Cell 2001, 33, 376–387. (31) Aizenberg, J.; Lambert, G.; Weiner, S.; Addadi, L. J. Am. Chem. Soc. 2002, 124, 32–39. (32) Rieger, J.; Frechen, T.; Cox, G.; Heckmann, W.; Schmidt, C.; Thieme, J. Faraday Discuss. 2007, 136, 265–277. (33) Pichon, B. P.; Bomans, P. H. H.; Frederik, P. M.; Sommerdijk, N. J. Am. Chem. Soc. 2008, 130, 4034–4040.

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Figure 1. SEM and optical micrographs of ACC films collected on a glass slide: (a) SEM image, (b) optical microscopy, (c) cross-polarized optical microscopy, and (d) cross-polarized optical microscopy with the first-order red λ-plate inserted. The films, seen as the same color as the background when using the first-order red λ-plate with cross-polarizers, indicates an isotropic feature.

50 mM CaCl2 concentration, ACC films can be formed within the polymer concentration range of 30-500 µg/mL, but the amount of ACC films collected varies. We thus chose an intermediate concentration (243.9 µg/mL) within this range to study the selforganization process. In control experiments without maleic chitosan, rhombohedral calcite crystals were collected after 40 min. Characterization. Fourier Transform Infrared Spectroscopy (FT-IR). Infrared (IR) spectroscopy was performed in a microscopycontinuum mode. Samples were analyzed using a Perkin-Elmer Spectrum One FT-IR spectrometer. Scanning Electron Microscopy (SEM). The morphology of the obtained samples was examined by scanning electron microscopy (KECK FE-SEM, LEO 1550 (Schottky field emitter)) after being sputter-coated with Au/Pd. The experiments were performed at a low accelerating voltage (3 KeV), especially suitable for imaging the surface detail of low-density materials. Polarized Optical Microscopy (POM). Fresh samples formed on glass slides or TEM grids were directly observed using a polarized optical microscope (Zeiss) equipped with cross-polarizers in the transmission mode. Some of these optical micrographs were taken with the insert of the first-order red λ-plate so that both birefringent and isotropic matter can be viewed in cross-polarized light. Transmission Electron Microscopy (TEM). Samples were directly formed on TEM carbon grids and then loaded using a single tilt holder. TEM was performed on a FEI Technai T12 instrument at 120 kV, and selected area electron diffraction (SAED) was recorded using the same equipment.

Results and Discussion The calcium carbonate precipitation was achieved using an ammonium carbonate diffusion method. Typical SEM morphology of ACCs formed at 30 min on a glass slide is shown in Figure 1a. It is a continuous film with a thickness less than 1 µm. The corresponding optical micrograph is shown in Figure 1b. The amorphous feature of the whole film was confirmed by polarized optical microscopy (Figure 1c and d). Figure 1d reveals that the films were seen as the same color as the background when the first-order red λ-plate with cross-polarizers was used, revealing an isotropic feature.19 The ACC characteristics of the structure are further confirmed by the FT-IR spectrum, which reveals the presence of a carbonate out-of-plane bending adsorption at 866 cm-1 (ν2), the symmetric stretch in the noncentrosymmetric structure expressed by the presence of a broad peak at 1080 cm-1 (ν1), and the splitting at 1420 and 1474 cm-1 of the peak due to the asymmetric stretch of the carbonate ion (Figure 2).1

Acid Polysaccharide-Induced ACC Films

Figure 2. FT-IR spectra of amorphous calcium carbonate film collected from the glass slide.

It is difficult to prepare very thin samples of ACC film for TEM characterization due to the rapid transformation of ACC into its crystalline phase during the microtome process. Accordingly, we decided to form ACC films directly onto TEM grids by floating the grids onto a crystallization solution. This approach also precludes artifacts produced during sample preparation via microtomy. ACC films can even be formed in the solution without the assistance of any substrate. Although the surface properties of glass slides and TEM grids are different, these differences did not affect the deposition of ACC films onto them. Figure 3 displays the morphology typical of the films deposited on TEM grids after 30 and 40 min. Moreover, they exhibit morphology similar to that of the films we obtained on glass slides (based on light microscopy and SEM images, as shown in Figures 1 and 3). The amorphous feature of both films is confirmed by cross-polarized optical microscopy (Figure 3c and Supporting Information Figure S2). After a deposition time of 40 min, continuous amorphous films with thicknesses about of 1-2 µm typically covered the whole TEM grid (Figure 3a and inset). In some cases, the films were not yet fully integrated, as indicated by the presence of boundaries between neighboring spherical particles with a size around 2 µm (Figure 3e). It is thus conceivable that the continuous integrated films formed through a gradual buildup of many small particles around 2 µm. The 30 min sample, examined from the deposition side, provides further insight into how the continuous films were formed. The whole film had a patterned morphology (Figure 3g). The formation of such a pattern apparently originates from the grid pattern due to the initial deposition of ACC phase along the rim, followed by gradual growth of more ACC phase toward the open area (Figure 3h). In addition, the ACC phase directly deposited on the rim exhibited a smooth surface due to full coalescence of the particles, whereas those close to the open areas had a rough surface, indicated by similar traces of boundaries as the 40 min sample, as a result of incomplete coalescence (Figure 3h). Accordingly, we believe that the films are formed through a gradual buildup of smaller particles (1-2 µm), with aggregation and coalescence occurring synchronously. This observation does not imply, however, that the 2 µm particles are necessarily the basic units for such aggregation and coalescence. Indeed, SEM images of higher magnification, shown in Figure 3f and i, reveal that these spherical particles are actually constructed from many smaller subunits around 100-200 nm. Our next step was to investigate the mesostructure of the ACC films using TEM. For the 40 min sample, the detailed structure was hard to detect due to the thickness of the films (Supporting Information Figure S3). However, we did find some thin area

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in the 30 min sample, composed of many particle clusters (Figure 4a). The size of each particle is around 100-200 nm, similar to the size of the particles we observed in the SEM image (Figure 3f, i). The amorphous feature of these nanoparticles was confirmed by the appearance of diffuse rings in the SAED pattern (Figure 4a inset). In some thinner areas, particularly those close to the film edges, even smaller particles were observed across sites (Figure 4b). Indeed, TEM of higher magnification (Figure 4c) reveals that these nanoparticles are less than 10 nm and tend to form a continuous but “disordered” nanofilm structure. It may be that the polysaccharide on their surfaces assists this aggregation. Similar observations can be extended to the film edges of different locations (Figure 4d, e). It is thus conceivable that these smallest particles are the basic units to construct the whole films. Interestingly, in some cases, individual mesopores with diameters ranging from 10 to 50 nm were sometimes observed to be embedded inside these nanoparticles (Figure 4f). It is not clear how such pores are formed, although the SAED pattern around the pore area also indicates its amorphous feature (Figure 4f inset). We suspect that the presence of these pores might be associated with colloidal nanoparticle self-assembly processes. There are reports in the literature that describe the formation of amorphous calcium carbonate films or crystalline films transforming via ACC films in the presence of acid polymer (poly(aspartic acid)).19,20 The capture of pure amorphous calcium carbonate films before the phase transformation was not achieved in the reports. It was however suggested that preformed polymerinduced liquid precursor (PILP) droplets (2-4 µm) settle and subsequently wet the substrate and spread into a film form.20 This suggestion was based on optical microscopy and SEM results. More recently, Rieger et al. have studied precursor structures in the precipitation processes of CaCO3 by mixing 0.01 M solutions of CaCl2 and Na2CO3 in the presence of polycarboxylic acid.32 They have found that, from an emulsion-like or phase-separated structure formed at the very beginning (obtained within 100 ms, and information about the chemical components is not known), ACC nanoparticles formed and then built into a floc structure, probably interconnected by the polymer coatings. Even in the absence of any additive, a short-lived filmlike structure could be formed and was believed to develop from spherical particles of different size. In contrast, we also observed filmlike structures in our studies even though we did not detect any emulsion-like structure even in the samples we collected at earlier stages (for example, 5-10 min samples). Moreover, our results seem to support the hypothesis that the films are formed through a gradual dynamic buildup of particles with different sizes (ranging from nanometers to micrometers), an inference based on the combined results from SEM, optical microscopy, and TEM. These films can even be formed around the open area of the TEM grid, which does not have a substrate to support its deposition (Figure 3a, d). Accordingly, we propose a colloidal nanoparticle selforganization model to explain the formation of continuous ACC films. This is illustrated in Scheme 1. At the initial stage, the acid polysaccharide molecules sequester and concentrate the ionic species and inhibit crystallization. It is likely that phase separation takes place at this stage. The resulting phase-separated structures might be highly hydrated and not easily collected under normal conditions. ACC nanoparticles of less than 10 nm then appear and are stabilized by acid polysaccharide molecules as local supersaturation increases. These ACC nanoparticles tend to aggregate to form nanoclusters and then self-organize into larger particles (100-200 nm), and these bigger particles, in turn, first aggregate to form bigger particle clusters and then self-organize

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Figure 3. SEM and crossed-polarized microscopy micrographs of ACC films directly deposited on TEM grids at different time intervals: (a-f) 40 min sample, (e) is the magnified picture of the selected area in (d) (indicated by white arrow), and the grain boundaries between particles are clearly seen (white arrows in (e)); (g-i) 30 min sample, examined from the deposition site. The ACC grows toward the open area even without the presence of a substrate (white arrow in (h)).

Figure 4. TEM pictures of 30 min ACC film sample directly deposited on TEM grids: (a) typical structure image of 30 min sample and ((a), inset) SAED pattern of 30 min sample, where the selected area is indicated by a white dot-dashed circle as shown in (a). (b) Typical structure image of 30 min sample examined at thin edges showing multiscale sizes of nanoparticles. (c) Detailed mesostructure around the thin edges showing nanoparticles of less than 10 nm. These nanoparticles aggregate to form the nanofilm structure. (d-f) more TEM images examined at different sites of thin edges of the 30 min sample. Similar mesoporous structures constructed from nanoparticles of less than 10 nm are found. ((f), inset) Corresponding SAED pattern of films shown in (f). The selective area is indicated by a white dot-dashed circle in (f).

into 2-3 µm particles. Further self-organization process between particles and/or particle clusters (including small/small particles or clusters, small/big particles or clusters, and big/big particles or clusters) finally leads to the formation of a continuous ACC film. It is interesting to note that the phenomenon of nanoparticle and nanocluster formation via aggregation has previously been observed in surfactant-stabilized ACC-containing reverse microemulsions34 and in several other solution systems including (34) Li, M.; Mann, S. AdV. Funct. Mater. 2002, 12, 773–779.

silica,35 aluminum ions,36 and other large cations.37 There is increasing evidence that nanocluster precursors in the aqueous phase may play an important role in crystallization and biomineralization.38 The process of self-organization associated with the general trend toward a minimal interfacial area (thus a minimal interfacial (35) Brinker, C. J.; Scherer, G. W. Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing; Academic: London, 1990. (36) Phillips, B. L.; Casey, W. H.; Karlsson, M. Nature 2000, 404, 379–382. (37) Casey, W. H.; Swaddle, T. W. ReV. Geophys. 2003, 41, 1–20. (38) Navrotsky, A. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12096–12101.

Acid Polysaccharide-Induced ACC Films Scheme 1. Proposed Colloidal Nanoparticle Self-Organization Model to Explain the Formation of Amorphous Films in the Presence of Acid Polysaccharide

energy) between the dispersed phase of small particles or particle clusters and the dispersion medium. This is primarily achieved by coagulation followed by coalescence. It is also possible that the functionalized surface of nanoparticles/nanoclusters assists in the self-organization, although very little is known about the surface at this point. Finally, the self-organization process obeys the Ostwald ripening process in which larger ACC colloidal particles grow at the expense of smaller ones due to particle diffusion through the continuous phase.

Conclusions We have demonstrated that ACC films can be formed in the presence of a novel synthetic acid polysaccharide template. We

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believe that this is the first report using an acid polysaccharide as an additive to prepare ACC films. By direct deposition of the ACC films onto TEM grids, we have successfully used TEM in combination with SAED to characterize the mesostructure of the formed ACC films. We propose a colloidal nanoparticle selforganization model to explain the formation of the continuous ACC films. Our studies indicate that polysaccharide macromolecules, particularly acid polysaccharides, are potential resources for producing stable ACC films. More generally, we believe that these several findings and our self-organization model provide insight into the formation of ACC films in the presence of additive/ template systems. Acknowledgment. The authors thank Professor Lara Estroff from Cornell’s Department of Materials Science for help with the crystallization setup and for providing insightful suggestions. This project was partially supported by graduate student thesis funds from the School of Human Ecology at Cornell University. This work made use of the electron microscopy (SEM and TEM) facilities of the Cornell Center for Materials Research (CCMR) with support from the National Science Foundation Materials Research Science and Engineering Centers (MRSEC) program (DMR 0520404). Supporting Information Available: Synthesis and chemical structure of maleic chitosan, additional optical and cross-polarized optical microscopy images of 40 min ACC sample, and TEM image of 40 min ACC sample. This material is available free of charge via the Internet at http://pubs.acs.org. LA803541M